Accurate chromosomal segregation is of fundamental importance for inheritance of genetic materials and cellular physiology. Altered chromosomal segregation often causes aneuploidy, a hallmark of human cancers. The central hypothesis of this proposal is that Cbx2, a member of the Cbx protein family of the PcG complex, controls chromosomal segregation by directly organizing mitotic chromosome topology and by coordinating with the chromosomal segregation proteins. The central hypothesis will be tested with two Specific Aims. Specific Aim 1 will define the role of Cbx2 in organizing mitotic chromosome topology and determine the mitosis-specific function of Cbx2 in chromosomal segregation and leukemogenesis. The working hypothesis for Specific Aim 1 is that the genome-wide occupancy of Cbx2 is distinct between interphase and mitosis, and the enrichment and immobilization of Cbx2 on mitotic chromosomes is critical for organizing chromosome architecture and for segregating mitotic chromosomes. The chromosome morphology will be analyzed in Cbx2 KO and wild-type ES cells. The genome-wide occupancy of Cbx2 in both interphase and mitosis will be determined by ChIP-Seq. The mitotic-specific roles of Cbx2 in chromosomal segregation and transcriptome regulation will be assayed by specifically degrading Cbx2 in mitosis without impacting its protein level in interphase. Since aneuploidy is a hallmark of acute myeloid leukemia and observed in MLL-AF9-induced leukemia, we will analyze the role of Cbx2 in MLL-AF9-induced leukemia. Specific Aim 2 will determine the physical and functional interdependence of Cbx2, Condensin and Sgo2 for chromosomal segregation. The working hypothesis for Specific Aim 2 is that Cbx2 coordinates with Condensin and Sgo2 to control chromosomal segregation. We will establish the physical interaction between Cbx2 and the chromosomal segregation proteins, and we will identify Cbx2 mutants that lack interaction with Condensin and Sgo2. We will determine the interdependence of Cbx2, Condensin and Sgo2 for localization on mitotic chromosomes and the functional interdependence of Cbx2, Condensin and Sgo2 for chromosomal segregation. The ability to identify and study entirely new pathways and functional roles for PcG protein Cbx2 will be of compelling interest to the fields of both transcription and chromosomal segregation. Since abnormal transcription and mis-segregation of chromosomes are the driving forces of cancer formation and progression, we are confident that these pathways will be targeted for developing new cancer treatment drugs.